Monoclonal and polyclonal antibodies are useful for a variety of purposes. The precise antigen specificity of antibodies makes them powerful tools that can be used for the detection, quantitation, purification and neutralization of antigens.
Polyclonal antibodies are produced in vivo by immunizing animals, such as rabbits and goats, with antigens, bleeding the animals and isolating polyclonal antibody molecules from the blood. Monoclonal antibodies are produced by hybridoma cells, which are made by fusing, in vitro, immortal plasmacytoma cells with antibody producing cells (Kohler, G. and C. Milstein, Nature, 256:495 (1975)) obtained from animals immunized in vivo with antigen.
Current methods for producing polyclonal and mono-clonal antibodies are limited by several factors. First, methods for producing either polyclonal or monoclonal antibodies require an in vivo immunization step. This can be time consuming and require large amounts of antigen. Second, the repertoire of antibodies expressed in vivo is restricted by physiological processes, such as those which mediate self-tolerance that disable-auto-reactive B cells (Goodnow, C. C., et al., Nature, 334:676 (1988); Goodnow, J. W., Basic and Clinical Immunology, Ed. 5, Los Altos, Calif., Large Medical Publications (1984); Young, C. R., Molecular Immunology, New York, Marcel Dekker (1984)). Third, although antibodies can exist in millions of different forms, each with its own unique binding site for antigen, antibody diversity is restricted by genetic mechanisms for generating antibody diversity (Honjo, T., Ann. Rev. Immunol., 1:499 (1983); Tonegawa, S., Nature:302:575 (1983)). Fourth, not all the antibody molecules which can be generated will be generated in a given animal. As a result, raising high affinity antibodies to a given antigen can be very time consuming and can often fail. Fifth, the production of human antibodies of desired specificity is very problematical.
A method of producing antibodies which avoids the limitations of presently-available methods, such as the requirement for immunization of an animal and in vivo steps, would be very useful, particularly if it made it possible to produce a wider range of antibody types than can be made using presently-available techniques and if it made it possible to produce human antibody types.
The present invention relates to a method of producing libraries of genes encoding antigen-combining molecules or antibodies; a method of producing antigen-combining molecules, also referred to as antibodies, which does not require an in vivo procedure, as is required by presently-available methods; a method of obtaining antigen-combining molecules (antibodies) of selected or defined specificity which does not require an in vivo procedure; vectors useful in the present method and antibodies produced or obtained by the method.
The present invention relates to an in vitro process for synthesizing DNA encoding families of antigen-combining molecules or proteins. In this process, DNA containing genes encoding antigen-combining molecules is obtained and combined with oligonucleotides which are homologous to regions of the genes which are conserved. Sequence-specific gene amplification is then carried out using the DNA containing genes encoding antigen-combining proteins as template and the homologous oligonucleotides as primers.
This invention also relates to a method of creating diverse libraries of DNAs encoding families of antigen-combining proteins by cloning the product of the in vitro process for synthesizing DNA, described in the preceeding paragraph, into an appropriate vector (e.g., a plasmid, viral or retroviral vector).
The subject invention provides an alternative method for the production of antigen-combining molecules, which are useful affinity reagents for the detection and neutralization of antigens and the delivery of molecules to antigenic sites. The claimed method differs from production of polyclonal antibody molecules derived by immunization of live animals and from production of mono-clonal antibody molecules through the use of hybridoma cell lines in that it does not require an in vivo immunization step, as do presently available methods. Rather, diverse libraries of genes which encode antigen-combining sites comprising a significant proportion of an animal""s repertoire of antibody combining sites are made, as described in detail herein. These genes are expressed in living cells, from which molecules of desired antigenic selectivity can be isolated and purified for various uses.
Antigen-combining molecules are produced by the present method in the following manner, which is described in greater detail below. Initially, a library of antibody genes which includes a set of variable regions encoding a large, diverse and random group of specificities derived from animal or human immunoglobulins is produced by amplifying or cloning diverse genomic fragments or cDNAs of antibody mRNAs found in antibody-producing tissue.
In an optional step, the diversity of the resulting libraries can be increased by means of random mutagenesis. The gene libraries are introduced into cultured host cells, which may be eukaryotic or prokaryotic, in which they are expressed. Genes encoding antibodies of desired antigenic specificity are identified, using a method described herein or known techniques, isolated and expressed in quantities in appropriate host cells, from which the encoded antibody can be purified.
Specifically, a library of genes encoding immunoglobulin heavy chain regions and a library of genes encoding immunoglobulin light chain regions are constructed. This is carried out by obtaining antibody-encoding DNA, which is either genomic fragments or cDNAs of antibody mRNAs, amplfying or cloning the fragments or cDNAs; and introducing them into a standard framework antibody gene vector, which is used to introduce the antibody-encoding DNA into cells in which the DNA is expressed. The vector includes a framework gene encoding a protein, such as a gene encoding an antibody heavy chain or an antibody light chain which can be of any origin (human, non-human) and can be derived from any of a number of existing DNAs encoding heavy chain immunoglobulins or light chain immunoglobulins. Such vectors are also a subject of the present invention and are described in greater detail in a subsequent section. Genes from one or both of the libraries are introduced into appropriate host cells, in which the genes are expressed, resulting in production of a wide variety of antigen-combining molecules.
Genes encoding antigen-combining molecules of desired specificity are identified by identifying cells producing antigen-combining molecules which react with a selected antigen and then obtaining the genes of interest. The genes of interest can subsequently be introduced into an appropriate host cell (or can be further modified and then introduced into an appropriate host cell) for further production of antigen-combining molecules, which can be purified and used for the same purposes for which conventionally-produced antibodies are used.
Through use of the method described, it is possible to produce antigen-combining molecules which are of wider diversity than are antibodies available as a result of known methods; novel antigen-combining molecules with a diverse range of specificities and affinities and antigen-combining molecules which are predominantly human in origin. Such antigen-combining molecules are a subject of the present invention and can be used clinically for diagnostic, therapeutic and prophylactic purposes, as well as in research contexts, and for other purposes.